Antibiotics produced by fermentation of a novel strain of Streptomyces hygroscopicus

ABSTRACT

Antibiotic Bu-2659 complex, containing components A, B, C, D and E, is produced by cultivation of Streptomyces hygroscopicus Strain No. J296-21, ATCC No. 39150.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a division of our prior, copending application Ser. No. 409,740, (now U.S. Pat. No. 4,468,386) filed Aug. 19, 1982.

SUMMARY OF THE INVENTION

This invention relates to novel antibiotic substances. More particularly, it relates to an antibiotic complex designated as Bu-2659, said complex being produced by cultivating a strain of Streptomyces hygroscopicus having the identifying characteristics of ATCC No. 39150 under submerged aerobic conditions in an aqueous nutrient medium until a substantial amount of Bu-2659 is produced in the culture medium and, optionally, recovering Bu-2659 from the culture medium.

This invention also provides five novel antibiotic components of Bu-2659, designated Bu-2659A, Bu-2659B, Bu-2659C, Bu-2659D and Bu-2659E, which are recovered from Bu-2659 complex by chromatographic procedures.

The Bu-2659 components A-E are structurally related to the neomycin, paromomycin and ribostamycin groups of antibiotics, but differ in that the aglycone of the Bu-2659 components is 1-deamino-1-hydroxy-2-deoxystreptamine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the infrared absorption spectrum of Bu-2659A when pelleted in KBr.

FIG. 2 shows the Proton Magnetic Resonance (PMR) spectrum of Bu-2659A in D₂ O (60 MHz).

FIG. 3 shows the Proton Magnetic Resonance (PMR) spectrum of Bu-2659B in D₂ O (60 MHz).

FIG. 4 shows the Proton Magnetic Resonance (PMR) spectrum of Bu-2659C in D₂ O (60 MHz).

FIG. 5 shows the Proton Magnetic Resonance (PMR) spectrum of Bu-2659D in D₂ O (60 MHz).

FIG. 6 shows the Proton Magnetic Resonance (PMR) spectrum of Bu-2659E in D₂ O (80 MHz).

DESCRIPTION OF THE PRIOR ART

There have been several recent examples of the production of aminoglycoside antibiotics containing the 1-deamino-1-hydroxy-2-deoxystreptamine moiety as the aglycone. Each of these were produced by fermentation of a 2-deoxystreptamine-negative mutant of a known microorganism. The preparation of Bu-2659 is the first instance of a 1-deamino-1-hydroxy-2-deoxystreptamine-containing aminoglycoside being produced by fermentation of a naturally occurring microorganism.

Published Japan Patent Application (Kokai) No. 54-117,477 [Chem. Abst. 92, 92700d (1980)] discloses antibiotic SUM-3, which is the 1-deamino-1-hydroxy analog of sagamicin. SUM-3 was produced by fermentation of Micromonospora sagamiensis SU-2 (FERM-P 4230, NRRL 11182) which is a 2-deoxystreptamine-negative mutant of the sagamicin-producing microorganism.

Published Japan Patent Application (Kokai) No. 55-99,196 [Chem. Abst. 94, 119467p (1981)] discloses antibiotics K-144e and K-144g, which are the 1-deamino-1-hydroxy analogs of gentamicin X₂ and G418, respectively. They also were produced by fermentation of Micromonospora sagamiensis SU-2.

The Journal of Antibiotics, 33, 836-841 (1980) discloses antibiotic S-11-A, which is the 1-deamino-1-hydroxy analog of xylostasin. It was produced by fermentation of Bacillus circulans S-11 (FERM-P 5220), a 2-deoxystreptamine-negative mutant of the xylostasin-producing microorganism Bacillus circulans Mot 3. The structures of Bu-2659E and S-11-A are closely related, the difference being in the pentose moiety. Bu-2659E contains ribose while S-11-A contains xylose.

The Journal of Antibiotics, 35, 520-523 (1982) discloses antibiotics SU-1, SU-2 and SU-3, which are the 1-deamino-1-hydroxy analogs of gentamicin C₂, gentamicin C_(1a) and sagamicin, respectively. They are prepared by fermentation of a 2-deoxystreptamine idiotrophic mutant of the sagamicin producer Micromonospora sagamiensis KY-11509.

COMPLETE DESCRIPTION

Bu-2659 complex and its components Bu-2659A, B, C, D and E may be produced by fermentation of Streptomyces hygroscopicus strain J296-21 (ATCC No. 39150). The structures of Bu-2659A, B, C, D and E (along with that of a bioactive degradation product of Bu-2659A designated as Bu-2659 DP-I) are as shown in Structures I and II below. The structures of neomycins B and C, paromomycins I and II, ribostamycin and neamine are also shown for comparison purposes.

    ______________________________________                                          ##STR1##                      I                                                                    R.sub.1                                                                              R.sub.2                                                                              R.sub.3 R.sub.4                               ______________________________________                                         Bu-2659A                       NH.sub.2                                                OH           H     CH.sub.2 NH.sub.2                                   Bu-2659B                       NH.sub.2                                                OH           CH.sub.2 NH.sub.2                                                                    H                                                   Bu-2659C                       OH                                                      OH           H     CH.sub.2 NH.sub.2                                   Bu-2659D                       NH.sub.2                                                OH           H     CH.sub.2 OH                                         neomycin B                     NH.sub.2                                                NH.sub.2     H     CH.sub.2 NH.sub.2                                   neomycin C                     NH.sub.2                                                NH.sub.2     CH.sub.2 NH.sub.2                                                                    H                                                   paromomycin I                  OH                                                      NH.sub.2     H     CH.sub.2 NH.sub.2                                   paromomycin II                 OH                                                      NH.sub.2     CH.sub.2 NH.sub.2                                                                    H                                                   ______________________________________                                          ##STR2##                      II                                                                      R.sub.1  R.sub.2                                       ______________________________________                                         Bu-2659E D-ribose              OH                                              ribostamycin                   NH.sub.2                                                 D-ribose                                                              Bu-2659 DP-I                   OH                                                       H                                                                     neamine  H                     NH.sub.2                                        ______________________________________                                    

Actinomycete Strain No. J296-21 was isolated from a soil sample collected in the Philippines. It has been deposited in the American Type Culture Collection, Washington, D.C., and added to its collection of microorganisms as ATCC No. 39150.

Strain No. J296-21 forms aerial and substrate mycelia, and the color of the aerial mycelium is white, later turning to gray. It forms coiled spore-chains on monopodially branched aerial sporophores, each containing 10 to 50 arthrospores in a chain. A tightly coiled spore-chain is often formed. The spores are oval in shape, 0.6-0.8 by 0.9-1.2 μm in size, and have a rugose or smooth surface. The spirals of the spore-chains often coalesce as dark, moist masses.

Strain J296-21 grows well and forms aerial mycelium in both nutritionally rich organic media and chemically defined agar media except for ISP No. 6 Medium. Strain J296-21 does not produce melanoid pigment in tryptone-yeast extract broth (ISP No. 1), peptone-yeast extract-iron agar (ISP No. 6) or tyrosine agar (ISP No. 7). It grows on agar medium containing NaCl at a concentration of 6% but not at 8%. Whorl sporophores, motile spores and sporangia were not observed in any of the media examined. The cultural and physiological characteristics of strain J296-21 are shown in Tables 1 and 2, respectively. The pattern of carbohydrate utilization is shown in Table 3.

The above-mentioned characteristics of strain J296-21 indicate that it belongs to the genus Streptomyces. According to the descriptions in Bergey's Manual of Determinative Bacteriology, 8th ed., strain J296-21 resembles the species group, spirales, gray series, non-chromogenic, and smooth spore suface, which includes 65 species and 7 subspecies. Hygroscopic change of the aerial mycelium (blackening and moistening) is an additional important property of strain J296-21. Dietz ["Criteria for Characterization of Hygroscopicus Strains" in Actinomycetes: The Boundary Microorganisms, edit. T. Arai, Toppan Co. Ltd., Japan, pp. 183-191 (1976)] classified hygroscopic Streptomyces strains into two species, S. hygroscopicus and S. neohygroscopicus. Based on the descriptions in the Bergey's Manual and the studies of Dietz, strain J296-21 was determined to belong to the species, Streptomyces hygroscopicus.

                  TABLE 1                                                          ______________________________________                                         Cultural Characteristics of Strain J296-21                                     ______________________________________                                         Czapek's sucrose-nitrate                                                                      G:    Abundant                                                  agar           R:    White (263) to light gray (264)                                          A:    Abundant, light gray (264) to                                                  medium gray (265)                                                        D:    None                                                      Tryptone-yeast extract                                                                        moderately floccose, sedimented,                                agar (ISP No. 1)                                                                              not pigmented                                                   Yeast extract-malt extract                                                                    G:    Abundant                                                  agar (ISP No. 2)                                                                              R:    Moderate orange yellow (71) to                                                 deep yellowish brown (75)                                                A:    Moderate, white (263)                                                    D:    None                                                      Oat meal agar (ISP No. 3)                                                                     G:    Abundant                                                                 R:    White (263) to light gray (264)                                          A:    Abundant, white (263) to medium                                                gray (265), hygroscopic                                                  D:    None                                                      Inorganic salts-starch                                                                        G:    Abundant                                                  agar (ISP No. 4)                                                                              R:    White (263) to light gray (264)                                          A:    Abundant, light gray (264) to                                                  dark gray (266), hygroscopic                                             D:    None                                                      Glycerol-asparagine agar                                                                      G:    Abundant                                                  (ISP No. 5)    R:    Light yellow (86) to strong                                                    yellow (84)                                                              A:    Abundant, white (263) to light                                                 brownish gray (63), hygroscopic                                          D:    Light greenish yellow (101)                               Peptone-yeast extract-                                                                        G:    Moderate                                                  iron agar (ISP No. 6)                                                                         R:    Pale yellow (89)                                                         A:    None                                                                     D:    None                                                      Tyrosine agar (ISP No. 7)                                                                     G:    Abundant                                                                 R:    Strong reddish brown (40)                                                A:    Abundant, white (263) to                                                       yellowish white (92)                                                     D:    Dark orange yellow (72)                                   Bennett's agar G:    Abundant                                                                 R:    Pale yellow (89) to dark                                                       yellow (88)                                                              A:    Abundant, white (263) to medium                                                gray (265), hygroscopic                                                  D:    None                                                      ______________________________________                                          Abbreviations                                                                  G: Growth                                                                      R: Reverse color                                                               A: Formation of aerial mycelium and aerial mass color                          D: Diffusible pigment                                                          Colors and numbers in parentheses follow the color standard described by       K. L. Kelly and D. B. Judd: ISCCNBS colorname charts illustrated with          centroid colors. U.S. Dept. of Comm. Circ. 553, Washington, D.C., Nov.,        1975.                                                                    

                                      TABLE 2                                      __________________________________________________________________________     Physiological Reactions                                                                             S. hygroscopicus                                          Test     Strain No. J296-21                                                                         (NRRLB-1340)                                                                               Methods and Materials                         __________________________________________________________________________     Nitrite from                                                                            Positive    Negative    Inorganic medium:                             nitrate                          Czapek's glucose                                                               nitrate broth.                                         Positive    Negative    Organic medium: 0.5%                                                           yeast extract, 1%                                                              glucose, 0.5% KNO.sub.3,                                                       0.1% CaCO.sub.3.                              Sodium chloride                                                                         Moderate growth at                                                                         Moderate growth at                                                                         Basal medium: 1% yeast                        tolerance                                                                               0.5% NaCl. Re-                                                                             1.5% NaCl. Re-                                                                             extract, 2% soluble                                    stricted growth at                                                                         stricted growth at                                                                         starch, 1.5% agar.                                     1.0-6.0% NaCl. No                                                                          8.0% NaCl.                                                         growth at 8% NaCl.                                                    Casein hydrolysis                                                                       Weakly positive (1-2                                                                       Positive (3-5 mm                                                                           Luedemann's agar                              in agar medium                                                                          mm hydrolyzed band                                                                         hydrolyzed band                                                                            medium.*                                               after 7 days).                                                                             after 7 days).                                            Reactions in                                                                            Not coagulated and                                                                         Not coagulated and                                        skimmed milk                                                                            completely pepton-                                                                         completely pepton-                                        solution ized.       ized.                                                     Gelatin stab                                                                            Liquefied   Liquefied                                                 Formation of                                                                            Negative    Negative    Tyrosine agar and                             melanoid                         peptone-yeast-iron                                                             agar and tryptone-                                                             yeast extract broth.                          Effect of                                                                               Maximal growth at 28-                                                                      Maximal growth at 28-                                                                      Yeast extract-malt                            temperature                                                                             37° C. Moderate                                                                     37° C. Moderate                                                                     extract agar.                                          growth at 20° C. and                                                                growth at 20° C. No                                         43° C. No growth at                                                                 growth at 5° C. and                                         5° C. and 45° C.                                                             43° C.                                             __________________________________________________________________________      *Leudemann, G. M., Intl. J. Syst. Bacteriol. 21: 240-247, 1971.          

                  TABLE 3                                                          ______________________________________                                         Carbohydrate Utilization                                                                             S. hygroscopicus                                                      Strain J296-21                                                                          NRRLB-1340                                               ______________________________________                                         Glycerol       +          +                                                    D(-)-Arabinose +          -                                                    L(+)-Arabinose +          +                                                    D-Xylose       +          +                                                    D-Ribose       +          +                                                    L-Rhamnose     +          -                                                    D-Glucose      +          +                                                    D-Galactose    +          +                                                    D-Fructose     +          +                                                    D-Mannose      +          +                                                    L(-)-Sorbose   -          -                                                    Sucrose        -          -                                                    Lactose        +          -                                                    Cellobiose     +          +                                                    Melibiose      +          -                                                    Trehalose      +          +                                                    Raffinose      +          -                                                    D(+)-Melezitose                                                                               -          -                                                    Soluble starch +          +                                                    Cellulose      +          -                                                    Dulcitol       -          -                                                    Inositol       +          -                                                    D-Mannitol     +          +                                                    D-Sorbitol     +          -                                                    Salicin        +          +                                                    ______________________________________                                          Basal medium: PridhamGottlieb's inorganic medium                         

Antibiotic complex Bu-2659 is produced by cultivating Streptomyces hygroscopicus Strain No. J296-21 under submerged aerobic conditions in an aqueous nutrient medium. The general procedures used for the culture of other actinomycetes are applicable to the cultivation of Streptomyces hygroscopicus Strain J296-21. The nutrient medium should contain one or more assimilable carbon sources such as glycerol, glucose, fructose, mannose, starch, dextrin, maltose, molasses, oils, fats and the like, either in purified or the crude state. The nutrient medium should also contain one or more assimilable nitrogen sources such as, for example, soybean meal, fish metal, malt extract, peptone, yeast extract, distiller's solubles, gluten meal, cornsteep liquor, cottonseed flour, casein, hydrolyzed protein substances, nitrates, ammonium salts, urea and the like. Nutrient inorganic salts such as sodium chloride, potassium phosphate, magnesium sulfate, calcium carbonate, and trace amounts of heavy metal salts such as copper, zinc, manganese, iron, and the like, may also be added to the medium. In the aerated submerged culture an antifoam such as liquid paraffin, soybean oil, fat or silicone may be utilized.

The fermentation temperature preferably should be in the range of from about 20° C. to about 43° C., and the most preferred range is from about 28° C. to about 37° C. The pH of the fermentation medium should be in the range of from about 5 to about 10, and the preferred range is from about 6 to about 8. Ordinarily, optimum antibiotic production is obtained in 4 to 7 days, during which the pH gradually rises to about 8-8.5.

When a tank fermentation is to be carried out, it is desirable to produce a vegetative inoculum in a nutrient broth by inoculating the broth culture with, for example, a slant culture or lyophilized culture of the organism. After obtaining an active inoculum in this manner, it is transferred aseptically to the fermentation tank medium. The antibiotic activity in the fermentation broth may be determined by the paper disc-agar diffusion method using Bacillus subtilis PCI 219 as the test organism.

After optimum broth potency (typically 300-350 mcg/mL) is obtained, the fermentation broth is filtered, preferably with filter aid. The mycelial cake usually is washed with water, and the combined filtrate and washings are then adjusted to a pH of about 7.0 and adsorbed on a column of resin such as Amberlite IRC-50 (NH₄ ⁺). It is developed with dilute NH₄ OH and the active eluate fractions are combined and concentrated. The concentrated Bu-2659 complex may then be separated into its components by chromatography on a resin such as Amberlite CG-50 (NH₄ ⁺), using increasing concentrations of dilute NH₄ OH for development. This procedure sometimes gives a mixture of components D and E. That mixture may, if desired, be separated by silica gel column chromatography using CH₃ OH--NH₄ OH--H₂ O as the solvent system.

Bu-2659 complex and the individual components thereof are usually obtained in their free base form, but may be converted to their acid addition salts by reaction with an acid in a conventional manner. This invention includes within its scope the pharmaceutically acceptable acid addition salts of Bu-2659 complex and its individual components. The pharmaceutically acceptable acid addition salts of Bu-2659 complex and its individual components include, for example, those obtained by reaction with an inorganic acid such as hydrochloric, hydrobromic, sulfuric, phosphoric, nitric or the like, as well as with an organic acid such as acetic, malic, citric, ascorbic, methanesulfonic or the like.

The sulfates of Bu-2659 components are freely soluble in water, slightly soluble in methanol and ethanol but practically insoluble in n-butanol, acetone and other organic solvents. They give positive reactions with ninhydrin and anthrone reagents, but are negative in the Tollens, Fehling and Sakaguchi reactions. The thin layer chromatograms (TLC) of the Bu-2659 components are shown in Table 4 compared with those of neomycin, paromomycin and ribostamycin.

                  TABLE 4                                                          ______________________________________                                         TLC of Bu-2659 A, B, C, D and E                                                Rf Value (by ninhydrin reagent)                                                                                 Paro- Ribo-                                   Sys-  Bu-2659            Neo-    mo-   sta-                                    tem*  A      B      C    D    E    mycin mycin mycin                           ______________________________________                                         S-110 0.31   0.35   0.39 0.43 0.43 0.23  0.33  0.33                            S-115S                                                                               0.47   0.54   0.54 0.63 0.60 0.40  0.51  0.52                            S-115A                                                                               --     --     --   0.57 0.06 --    --    0.11                            ______________________________________                                          *S-110: SiO.sub.2 plate, CHCl.sub.3 --CH.sub.3 OH--conc. NH.sub.4              OHH.sub.2 O (1:4:2:1)                                                          S115S: SiO.sub.2 plate, CHCl.sub.3 --CH.sub.3 OH--17% NH.sub.4 OH (2:1:1:      upper layer)                                                                   S115A: Al.sub.2 O.sub.3 plate, solvent system same as S115S              

The physico-chemical properties of the sulfates of Bu-2659A, B, C, D and E are summarized in Table 5. The Bu-2659 components exhibit only end absorption in the UV spectra. The IR spectrum of Bu-2659A (FIG. 1) is very similar to that of components B, C, D and E, showing characteristics of the spectra of aminoglycoside antibiotics such as neomycin, paromomycin and ribostamycin. The proton NMR spectra of Bu-2659 components A, B, C, D and E are shown in FIGS. 2 through 6, respectively. The presence of three anomeric protons was indicated in the NMR spectra of Bu-2659 components A, B, C and D, while two anomeric protons were observed in the spectrum of component E (Table 5).

                                      TABLE 5                                      __________________________________________________________________________     Physico-chemical Properties of Bu-2659 Components (Sulfates)                           Bu-2659A Bu-2659B Bu-2659C Bu-2659D Bu-2659E                           __________________________________________________________________________     Nature  white amorphous                                                                         white amorphous                                                                         white amorphous                                                                         white amorphous                                                                         white amorphous                            solid    solid    solid    solid    solid                              Mp (°C.)                                                                        210˜215 (dec.)                                                                    200˜206 (dec.)                                                                    195˜201 (dec.)                                                                    193˜204 (dec.)                                                                    195˜218 (dec.)               [α].sub.D (H.sub.2 O)**                                                          +52° (c = 0.6)                                                                   +73° (c = 0.6)                                                                   +55° (c = 0.6)                                                                   +52.5° (c = 0.6)                                                                 +47° (c = 0.37)             Molecular                                                                              C.sub.23 H.sub.45 N.sub.5 O.sub.14                                                      C.sub.23 H.sub.45 N.sub.5 O.sub.14                                                      C.sub.23 H.sub.44 N.sub.4 O.sub.15                                                      C.sub.23 H.sub.44 N.sub.4 O.sub.15                                                      C.sub.17 H.sub.33 N.sub.3                                                      O.sub.11                           formula                                                                        Elemental                                                                              C.sub.23 H.sub.45 N.sub.5 O.sub.14.                                                     C.sub.23 H.sub.45 N.sub.5 O.sub.14.                                                     C.sub.23 H.sub.44 N.sub.4 O.sub.15.                                                     C.sub.23 H.sub.44 N.sub.4 O.sub.15.                                                     C.sub.17 H.sub.33 N.sub.3                                                      O.sub.11.                          analysis                                                                               2.5H.sub.2 SO.sub.4.2H.sub.2 O                                                          2.5H.sub.2 SO.sub.4.2.5H.sub. 2 O                                                       2H.sub.2 SO.sub.4.3H.sub.2 O                                                            2H.sub.2 SO.sub.4 -3H.sub.2 O                                                           1.5H.sub.2 SO.sub.4.2.5H.sub.2                                                  O                                         Calc'd                                                                             Found                                                                               Calc'd                                                                             Found                                                                               Calc'd                                                                             Found                                                                               Calc'd                                                                             Found                                                                               Calc'd                                                                              Found                         C %     30.81                                                                              30.94                                                                               30.50                                                                              30.57                                                                               31.87                                                                              31.89                                                                               31.87                                                                              31.50                                                                               31.53                                                                               31.24                         H %     6.07                                                                               6.62 6.12                                                                               6.35 6.28                                                                               6.29 6.28                                                                               6.15 6.38 6.34                          N %     7.81                                                                               8.01 7.73                                                                               7.52 6.46                                                                               6.27 6.46                                                                               6.83 6.49 6.60                          S %     8.92                                                                               9.23 8.83                                                                               8.68 7.40                                                                               7.46 7.40                                                                               7.79 7.43 7.65                          NMR spec-                                                                              1.5˜2.7 (2H,m)                                                                    1.4˜2.7 (2H,m)                                                                    1.5˜2.7 (2H,m)                                                                    1.5˜2.6 (2H,m)                                                                    2.14 (1H,q,J=12)                   trum* (δ, ppm)                                                                   3.1˜4.7 (22H,m)                                                                   3.1˜4.7 (22H,m)                                                                   3.2˜4.8 (22H,m)                                                                   3.0˜4.7 (22H,m)                                                                   2.75 (1H,dt,J=12,4.0)                      5.35 (1H,br.s)                                                                          5.40 (2H,br.s)                                                                          5.35 (1H,br.s)                                                                          5.25 (H,br.s)                                                                           3.5˜4.7 (16H,m)                      5.47 (1H,br.s)                                                                          5.95 (1H,d,J = 3.6)                                                                     5.40 (1H,br.s)                                                                          5.42 (1H,br.s)                                                                          5.76 (1H,s)                                6.09 (1H,d,J=3.6) 5.80 (1H,d,J=3.6)                                                                       6.02 (1H,d,J=4.0)                                                                       6.43 (1H,d,J                       __________________________________________________________________________                                                 =4.0)                               *60 MHz in D.sub.2 O, PD <  2.0 for Bu2659A, B, C and D. 80 MHz in D.sub.      O at PD 4.6 for Bu2659E. TMS was used as external standard. Abbreviations      s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br.: broad,      dt: double triplet. Coupling constant (J) given in Hz.                         **Temperature of [α].sub.D determination: A 27° C.; B             25° C.; C 25° C.; D 25° C., E 26° C.         

Bu-2659A (1.56 g) was hydrolyzed with 0.4N methanolic hydrogen chloride (455 mL) at reflux temperature for 4 hours. The hydrolyzate was chromatographed on a column of CG-50 (NH₄ ⁺, 360 mL) to afford a bioactive degradation product designated as Bu-2659 DP-I (657 mg). The structure of this compound has been determined to be 1-deamino-1-hydroxyneamine (see Formula II). The TLC of Bu-2659 DP-I is shown in Table 6 compared with that of neamine.

                  TABLE 6                                                          ______________________________________                                         TLC of Bu-2659 DP-I and Neamine                                                            Rf Value (by ninhydrin)                                            System*       Bu-2659 DP-I                                                                              Neamine                                               ______________________________________                                         S-110         0.55       0.48                                                  S-108         0.47       0.32                                                  S-123         0.48       0.24                                                  ______________________________________                                          *S-108: SiO.sub.2 plate, AcetoneAcOH--H.sub.2 O (20:6:74)                      S123: SiO.sub.2 plate, 10% AcONH.sub.4 --CH.sub.3 OH--10% NH.sub.4 OH          (9:10:1)                                                                 

The minimum inhibitory concentrations (MIC) of Bu-2659A, B, C, D and E were determined for a variety of gram-positive and gram-negative bacteria by the serial two-fold agar dilution method using the Steer's multi-inoculating apparatus. The inoculum was standardized as a 10⁴ dilution of an overnight culture of the test organisms in Heart Infusion Broth (Difco). Mueller-Hinton agar medium (Difco) was generally used for the MIC determination for most test organisms, GC medium (Eiken) for fastidious bacteria such as streptococci, Neisseria and Haemophilus species, and No. 1001 agar medium [3% glycerol, 0.3% sodium L-glutamate, 0.2% peptone, 0.31% Na₂ HPO₄, 0.1% KH₂ PO₄, 0.005% ammonium citrate, 0.001% MgSO₄, 1.5% agar] for mycobacteria. Reference antibiotics utilized in these tests included commercially available neomycin as well as its components, neomycins B and C [as used herein, the term "neomycin" means the commercial product containing neomycins B and C at an approximate ratio of 7:3], paromomycin [the commercial product containing >90% of paromomycin I and <10% of paromomycin II] and ribostamycin.

The in vitro antibacterial spectra of the Bu-2659 components are shown in Table 7. Bu-2659A showed the highest activity among the five Bu-2659 components, its activity being comparable to that of neomycin. Bu-2659B and C were approximately 1/4 as active as Bu-2659A, while Bu-2659D and E were less active than components B and C. It was surprising to find that Bu-2659A has substantially the same level of antibacterial activity as neomycin B (which is the 2-deoxystreptamine-containing congener or Bu-2659A, since the 1-deamino-1-hydroxy analogs of aminoglycoside antibiotics reported to date are generally less active than their parent antibiotics.

Bu-2659 complex and its components A, B, C, D and E are antibiotics which are useful in the treatment of bacterial infections in mammals, including man. They may be utilized orally, parenterally or topically, and may be administered alone or in conjunction with other active ingredients. The preferred component is Bu-2659A. It may be administered in the amounts and the dosage forms commonly utilized for neomycin. Components B, C, D and E may be utilized in the same dosage forms and are used in amounts appropriate to their bioactivity.

                                      TABLE 7                                      __________________________________________________________________________     Antibacterial Activities of Bu-2659 Components                                                MIC (mcg/mL)                                                                   Bu-2659                  Neomycin     Paromo-                                                                             Ribosta-             Test Organism  A  B     C     D    E    *   B   C    mycin                                                                               mycin                __________________________________________________________________________     Staphylococcus aureus 209P                                                                    0.8                                                                               3.1   3.1   6.3  25   0.4 0.4 0.8  0.8  1.6                  Staphylococcus aureus Smith                                                                   1.6                                                                               3.1   6.3   25   50   0.8 0.4 1.6  1.6  3.1                  Staphylococcus aureus D136                                                                    0.8                                                                               6.3   6.3   25   50   0.8 0.4 1.6  0.8  3.1                  Micrococcus luteus PCI 1001                                                                   3.1                                                                               >100  >100  100  >100 0.8 0.8 50   6.3  6.3                  Micrococcus flavus D12                                                                        6.3                                                                               >100  >100  100  >100 3.1 1.6 100  12.5 12.5                 Bacillus subtilis PCI 219                                                                     0.1                                                                               0.4   0.8   1.6  3.1  <0.05                                                                              <0.05                                                                              <0.05                                                                               0.2  0.4                  Escherichia coli NIHJ                                                                         1.6                                                                               12.5  6.3   12.5 50   1.6 1.6 3.1  3.1  3.1                  Escherichia coli Juhl                                                                         3.1                                                                               50    25    50   >100 3.1 3.1 12.5 12.5 6.3                  Klebsiella pneumoniae D11                                                                     0.2                                                                               1.6   1.6   1.6  12.5 0.4 0.2 0.4  0.4  0.4                  Proteus vulgaris A9436                                                                        0.4                                                                               3.1   1.6   3.1  12.5 0.8 0.8 1.6  0.8  0.8                  Proteus mirabilis A9554                                                                       0.8                                                                               6.3   3.1   6.3  12.5 0.8 0.8 3.1  0.8  1.6                  Proteus mirabilis A9906                                                                       0.8                                                                               6.3   3.1   6.3  25   0.8 0.8 1.6  0.8  1.6                  Proteus morganii A9553                                                                        1.6                                                                               12.5  6.3   12.5 25   3.1 3.1 3.1  1.6  1.6                  Proteus rettgeri A15167                                                                       0.8                                                                               3.1   3.1   3.1  12.5 1.6 1.6 1.6  1.6  0.8                  Pseudomonas aeruginosa D15                                                                    3.1                                                                               >100  25    >100 >100 3.1 3.1 >100 50   >100                 Pseudomonas aeruginosa A9930                                                                  1.6                                                                               >100  12.5  50   >100 3.1 3.1 50   12.5 100                  Serratia marcescens A20019                                                                    3.1                                                                               100   6.3   100  >100 1.6 1.6 6.3  1.6  25                   __________________________________________________________________________      *commercial product (neomycin B:C = ca. 7:3)?                            

The antibacterial activity of Bu-2659A against strains of Streptococcus, Neisseria, Haemophilus and Mycobacterium is shown in Table 8. Bu-2659A and neomycin showed equivalent activity against the species of Streptococcus and Mycobacterium, while Bu-2659A was more active than neomycin against Neisseria and Haemophilus species.

                  TABLE 8                                                          ______________________________________                                         Antibacterial Activities of Bu-2659 A                                                          MIC (mcg/mL)                                                                                        Neo-                                      Test Organisms    Media    Bu-2659 A mycin                                     ______________________________________                                         Streptococcus pyogenes A20201                                                                    GC       25        12.5                                      Streptococcus pneumoniae Type I                                                                  "        25        25   Neisseria gonorrhoeae                                                     A20143 " 50 >400                          Neisseria meningitidis A21496                                                                    "        50         400                                      Haemophilus influenzae A9729                                                                     "        50         400                                      Mycobacterium smegmatis 607                                                                      No. 1001 0.4       0.4                                       Mycobacterium phlei                                                                              "        0.1       0.2                                       Mycobacterium ranae                                                                              "        0.4       0.4                                       ______________________________________                                    

A series of aminoglycoside resistant organisms which have been shown to produce aminoglycoside-modifying enzymes were examined for their susceptibility toward Bu-2659A and reference antibiotics. The results are shown in Table 9. Bu-2659A was more active than neomycin, paromomycin and ribostamycin against several resistant strains which produce APH(3') or AAC(3).

                                      TABLE 9                                      __________________________________________________________________________     Activities of Bu-2659A and Reference Antibiotics                               Against Aminoglycoside-Resistant Organisms                                     Test      Inactivating**                                                                        MIC* (mcg/mL)                                                 Organism  Enzyme Bu-2659A                                                                             Neomycin                                                                             Paromomycin                                                                           Ribostamycin                               __________________________________________________________________________     Staphylococcus                                                                           APH(3')-I,II                                                                          12.5  25    >100   >100                                       aureus A20239                                                                  Bacillus brevis                                                                          ANT (4')                                                                              >100  25    >100   >100                                       IFO 12334                                                                      Escherichia coli                                                                         APH(3')-I                                                                             >100  >100  >100   >100                                       ML 1630                                                                        Escherichia coli                                                                         APH(3')-II                                                                            25    100   >100   >100                                       A20107                                                                         Escherichia coli                                                                         APH(3')-II                                                                            >100  >100  >100   >100                                       JR66/W677 ANT(2")                                                              Escherichia coli                                                                         AAC(3)-I                                                                              1.6   1.6   1.6    1.6                                        JR88                                                                           Escherichia coli                                                                         APH(3')-I                                                                             25    >100  >100   >100                                       JR35/C600                                                                      Escherichia coli                                                                         ANT(2")                                                                               0.8   0.8   1.6    1.6                                        A20732                                                                         Escherichia coli                                                                         AAC(6')-I                                                                             50    50    >100   >100                                       NR79/W677                                                                      Enterobacter                                                                             APH(3')-I                                                                             50    >100  >100   >100                                       cloacae A20364                                                                 Pseudomonas aerug-                                                                       AAC(3)-I                                                                              6.3   12.5  >100   >100                                       inosa A20601                                                                             APH(3')-II                                                           Pseudomonas aerug-                                                                       AAC(3)-II                                                                             >100  >100  > 100  >100                                       inosa  A20896                                                                  __________________________________________________________________________      *Mueller-Hinton agar (Difco)                                                   **APH: aminoglycoside phosphotransferase                                       ANT: aminoglycoside nucleotidyltransferase                                     AAC: aminoglycoside acetyltransferase                                    

The in vitro activity of Bu-2659 DP-I (1-deamino-1-hydroxyneamine) is shown in Table 10 along with that of neamine. In contrast to the structure-activity relationship observed with Bu-2659A and neomycin, the degradation product of Bu-2659A was much less active than neamine, the corresponding bioactive fragment derived from neomycin.

                  TABLE 10                                                         ______________________________________                                         Antibacterial Spectrum of Bu-2659 DP-I and Neamine                                              MIC (mcg/mL)*                                                 Test Organisms     Bu-2659 DP-I                                                                              Neamine                                          ______________________________________                                         Staphylococcus aureus 209P                                                                        200        6.3                                              Staphylococcus aureus Smith                                                                       200        3.1                                              Bacillus subtilis PCI 219                                                                         100        3.1                                              Escherichia coli NIHJ                                                                             200        12.5                                             Escherichia coli JR88                                                                             200        12.5                                             Klebsiella pneumoniae D11                                                                         200        12.5                                             Proteus vulgaris A9436                                                                            200        6.3                                              Proteus mirabilis A9554                                                                           800        25                                               Pseudomonas aeruginosa A9930                                                                      >800       400                                              ______________________________________                                          *Mueller-Hintno agar                                                     

The in vivo activity of Bu-2659A was assessed by experimental systemic infection in mice. The pathogenic bacteria used in the in vivo tests were E. coli Juhl, K. pneumoniae D-11, P. mirabilis A9906, S. aureus Smith and S. pyogenes A20201. Mice were challenged intraperitoneally with approximately 100×LD₅₀ dose of the pathogens in a 5% suspension of hog gastric mucin (American Laboratories, Omaha, Neb.). A single intramuscular treatment with the antibiotic was given immediately after the bacterial challenge. Five mice were used for each dosage level and the animals were observed for 4 days to determine the median protective dose (PD₅₀). Neomycin was comparatively tested as a reference compound. As shown in Table 11, Bu-2659A afforded excellent protection in mice against the five experimental infections tested. Bu-2659A showed in vivo activity comparable to that of neomycin against infections with three gram-negative bacteria. In terms of PD₅₀ value Bu-2659A was about one-half as active as neomycin against infections with S. aureus and S. pyogenes.

                  TABLE 11                                                         ______________________________________                                         In vivo Activity of Bu-2659 A                                                                   PD.sub.50 (mg/kg, im)                                         Test Organisms     Bu-2659 A Neomycin                                          ______________________________________                                         Escherichia coli Juhl                                                                             3.1       3.1                                               Klebsiella pneumoniae D-11                                                                        1.1       0.9                                               Proteus mirabilis A9906                                                                           2.5       1.8                                               Staphylococcus aureus Smith                                                                       0.63      0.31                                              Streptococcus pyogenes A20201                                                                     50        50                                                ______________________________________                                    

The acute toxicity of Bu-2659A was determined in mice by intravenous, intraperitoneal and subcutaneous routes. Neomycin also was tested for comparison purposes. As shown in Table 12, Bu-2659A was significantly less toxic than neomycin by all routes of administration. The toxicity of Bu-2659A was approximately 1/3 that of neomycin in terms of LD₅₀ values, thus presenting a potential clinical advantage for Bu-2659A.

                  TABLE 12                                                         ______________________________________                                         Acute Toxicities of Bu-2659 A                                                  LD.sub.50 (mg/kg)                                                              intravenous     intraperitoneal                                                                            subcutaneous                                       ______________________________________                                         Bu-2659 A                                                                              110         460         720                                            Neomycin                                                                                34         180         280                                            ______________________________________                                    

EXAMPLE 1 Preparation of Seed Culture and Small Scale Shake Flask Fermentations

A well-grown agar slant culture of the Bu-2659 producing organism, Strain No. J296-21, was inoculated into a 500-mL Erlenmeyer flask containing 100 mL of seed medium composed of malt extract 2%, fish meal 2%, peptone 0.2%, NaCl 0.5%, CaCO₃ 0.4% and MgSO₄ 0.05%. The pH was adjusted to 7.2 before sterilization. The seed culture was incubated at 28° C. for 96 hours on a rotary shaker (250 rpm), and 3 mL of the growth was transferred to 500-mL Erlenmeyer flasks containing 100 mL of production medium having the same composition as the seed medium. Upon shaking fermentation at 28° C., the broth pH gradually rose to 8.0-8.4 after 4-7 days and the antibiotic production reached a maximum of 300˜350 mcg/mL. The antibiotic activity in the fermentation broth was determined by the paper disc-agar diffusion method using Bacillus subtilis PCI 219 as a test organism.

EXAMPLE 2 Isolation and Purification

A combined fermentation broth from shake flasks (32 liters, 300 mcg/mL) was filtered with filter aid and the filtrate was adjusted to pH 7.0 with 6N HCl. The antibacterial activity in the filtrate was adsorbed on a column of Amberlite IRC-50 (NH₄ ⁺, 1.6 L). The column was washed with water (5 L) and then developed with 0.5N NH₄ OH solution (4 L). The active eluates were combined and concentrated in vacuo to a small volume, and the concentrate was chromatographed using a column of Amberlite CG-50 (NH₄ ⁺, 1.0 L). The column was washed with water (2 L) and then developed with increasing concentrations of aqueous ammonia. A mixture of components B, C, D and E was eluted first with 0.21N NH₄ OH (3 L) followed by component A which was eluted with 0.43N NH₄ OH (4 L). The mixture of components B, C, D and E was separated by chromatography on Amberlite CG-50 (NH₄ ⁺, 200 mL) column. A mixture of components D and E was eluted first with 0.1N NH₄ OH (0.5 L), then components B and C were eluted successively with 0.167N (0.7 L) and 0.21N (1.0 L) NH₄ OH solutions, respectively. The mixture of components D and E was further separated into each component by silica gel column chromatography (Wakogel C-200, 40 g) using a solvent system of CH₃ OH-conc. NH₄ OH--H₂ O (9:1:1). Yields for components A, B, C, D and E in the above experiment were 7,100 mg, 370 mg, 350 mg, 40 mg and 20 mg, respectively.

The free base of Bu-2659A (1,150 mg) was dissolved in a small amount of water and the pH adjusted to 4.5 with diluted H₂ SO₄. Addition of methanol to the solution yielded a white precipitate which was collected by filtration and dried in vacuo to give the sulfate of Bu-2659A (1,280 mg). The sulfates of other components were prepared in the same manner. The physico-chemical properties of Bu-2659A, B, C, D and E produced in this example are set forth in Table 5.

The structures of Bu-2659 components A, B, C, D and E are as shown in Formulae I and II above. For convenience, names of the components which show their relationship to known antibiotics are set forth below.

Bu-2659A 1-deamino-1-hydroxyneomycin B

Bu-2659B 1-deamino-1-hydroxyneomycin C

Bu-2659C 1-deamino-1-hydroxyparomomycin I

Bu-2659D 1,6'"-dideamino-1,6'"-dihydroxyneomycin B

Bu-2659E 1-deamino-1-hydroxyribostamycin 

We claim:
 1. A process for the preparation of antibiotic Bu-2659 complex which comprises cultivating a strain of Streptomyces hygroscopicus ATCC No. 39150 under submerged aerobic conditions in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen at a temperature of from about 20° C. to about 43° C. until a substantial amount of Bu-2659 complex is produced and accumulated in the culture medium, and recovering the Bu-2659 complex from the culture medium.
 2. The process of claim 1 which includes the additional step of separating Bu-2659 complex into components A, B, C, D and E by chromatographic means.
 3. The process of claim 3 wherein the cultivation is conducted at a temperature of from about 28° C. to about 37° C.
 4. A biologically pure culture of the Bu-2659 complex-producing microorganism Streptomyces hygroscopicus Strain No. J296-21, ATCC No.
 39150. 